YM155在肿瘤细胞化疗增敏中的作用及机制研究

发布时间：2019-05-30 11:48

【摘要】：背景:肿瘤是一种世界范围内均很常见的多发病,而恶性肿瘤则是目前对人类健康的危害表现最严重的一类疾病。我国最常为人所知和危害国人最严重的肿瘤有肺癌、乳腺癌、胃癌、肝癌、宫颈癌、白血病等,其中肺癌是最常见的、死亡率最高的肿瘤,而乳腺癌是中国女性所患最常见的恶性肿瘤。课题组前期研究发现凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族成员survivin,其蛋白水平在阿霉素耐药及高转移性乳腺癌细胞中表达上调,提示survivin在乳腺癌生存和转移中发挥了重要作用。YM155,survivin靶向抑制剂,其能显著下调乳腺癌细胞中survivin的表达。因此,我们推测YM155可能通过抑制survivin来促进肿瘤细胞凋亡,增加肿瘤细胞对化疗药物的敏感性,在肿瘤细胞的生存及恶性肿瘤的治疗中起到重要作用。目的:通过体外细胞学等实验检测YM155对4种不同生物学特性的肿瘤细胞系(2种肺癌细胞及2种乳腺癌细胞)的促凋亡作用,以及检测YM155是否增强肿瘤细胞对化疗药物紫杉醇(Paclitaxel,PAC)的敏感性,并阐述不同肿瘤细胞对YM155敏感性不同的可能原因及其机制。方法:1.采用体外实验如MTT、克隆形成等实验检测YM155及紫杉醇单独或联合使用时对4种肿瘤细胞的增殖所产生的影响;2.采用体外实验如MTT、克隆形成、细胞凋亡、细胞周期等实验检测不同浓度的YM155作用于4种肿瘤细胞时对其增殖、凋亡、周期所产生的影响,并观察4种肿瘤细胞对YM155敏感性是否不同;3.采用体外实验如western blot,q RT-PCR等实验检测P53,survivin,NF-κB通路等指标的变化,分析YM155促凋亡及其敏感性差异的作用机制。结果:1.YM155与化疗药物紫杉醇均抑制肿瘤细胞增殖诱导其凋亡,而YM155与紫杉醇联合使用时所表现出的促进凋亡作用明显优于单独使用紫杉醇;2.YM155对肿瘤细胞的杀伤作用呈现浓度依赖性,随着浓度的增加对肿瘤细胞的杀伤作用也增加。并且p53状态不同的肿瘤细胞对YM155的敏感性也不同,p53野生型的肿瘤细胞对YM155的敏感性要低于p53突变型或p53缺失型的肿瘤细胞;3.P53状态不同的肿瘤细胞对YM155所产生的诱导MDM2的作用并不相同。在低浓度YM155的刺激下,P53野生型肿瘤细胞中MDM2转录水平上升,YM155作为survivin的靶向抑制剂仍可特异性降低survivin的表达。结论:1.YM155可增加肿瘤细胞对化疗药紫杉醇的敏感性2.p53状态不同的肿瘤细胞对YM155的敏感性不同3.YM155作用的差异性是因为p53依赖的MDM2调控
[Abstract]:Background: tumor is a common disease in the world, and malignant tumor is the most serious disease to human health. The most commonly known and harmful tumors in China are lung cancer, breast cancer, gastric cancer, liver cancer, cervical cancer, leukemia and so on. Among them, lung cancer is the most common and the highest mortality tumor. Breast cancer is the most common malignant tumor in Chinese women. The previous study of the research group found that the protein level of survivin, a member of the apoptosis inhibitor protein (inhibitor of apoptosis protein,IAP family, was up-regulated in adriamycin resistant and highly metastatic breast cancer cells. It is suggested that survivin plays an important role in the survival and metastasis of breast cancer. YM155, survivin targeted inhibitor can significantly down-regulate the expression of survivin in breast cancer cells. Therefore, we speculate that YM155 may play an important role in the survival of tumor cells and the treatment of malignant tumors by inhibiting survivin to promote apoptosis of tumor cells and increase the sensitivity of tumor cells to chemotherapeutic drugs. Objective: to detect the apoptosis-promoting effect of YM155 on four tumor cell lines (2 kinds of lung cancer cells and 2 kinds of breast cancer cells) with different biological characteristics. To detect whether YM155 enhances the sensitivity of tumor cells to paclitaxel (Paclitaxel,PAC), and to elucidate the possible causes and mechanisms of different sensitivity of different tumor cells to YM155. Method: 1. The effects of YM155 and paclitaxel on the proliferation of four kinds of tumor cells were detected by in vitro experiments such as MTT, clone formation. 2. In vitro experiments, such as MTT, clone formation, apoptosis, cell cycle and so on, were used to detect the effects of different concentrations of YM155 on the proliferation, apoptosis and cycle of four kinds of tumor cells. The sensitivity of the four tumor cells to YM155 was observed. 3. The changes of P53, survived, NF-魏 B pathway and other indexes were detected by in vitro experiments such as western blot,q RT-PCR, and the mechanism of YM155 promoting apoptosis and its sensitivity was analyzed. Results: both 1.YM155 and paclitaxel inhibited the proliferation of tumor cells and induced apoptosis. The effect of YM155 combined with paclitaxel on promoting apoptosis was significantly better than that of paclitaxel alone. The killing effect of 2.YM155 on tumor cells was concentration-dependent, and the killing effect on tumor cells also increased with the increase of concentration. The sensitivity of tumor cells with different p53 status to YM155 was also different. The sensitivity of p53 wild type tumor cells to YM155 was lower than that of p53 mutant or p53 deficient tumor cells. The effect of tumor cells with different 3.P53 states on the induction of MDM2 produced by YM155 is not the same. Under the stimulation of low concentration of YM155, the transcription level of MDM2 in p53 wild type tumor cells increased. YM155, as a target inhibitor of survivin, could specifically decrease the expression of survivin. Conclusion: 1.YM155 can increase the sensitivity of tumor cells to paclitaxel. The difference of sensitivity of tumor cells to YM155 in different state of 2.p53 is due to the regulation of p53 dependent MDM2.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.53

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